A method for an assay of an activator or an inhibitor to an enzyme generally comprises mixing the enzyme, a substrate of the enzyme, anything essential to an enzyme reaction such as a metal ion and a test substance, reacting the mixture under a condition where the enzyme reaction can be carried out such as a buffer condition, a reaction temperature or the like, measuring or calculating an enzyme activity by any means and comparing it with an enzyme activity in the absence of a test substance.
For example, an assay of an activator or an inhibitor to a polymerase can be carried out as follows. First, a polymerase, a labeled substrate, a template, a primer, a metal ion and a test substance are mixed at the same time or in order for the polymerase reaction. The labeled substrate is incorporated to a reaction product, which is known as an incorporation. After reaction, an amount of the labeled substrate incorporated to the reaction product is measured. An enzyme activity calculated on the basis of the above measured amount is compared with that in the absence of a test substance.
Even if a result of the above assay suggests that a test substance may possess an activity or inhibitory activity to an enzyme, the activity may be pseudo-positive due to nonspecifically acting or binding to the enzyme based on the structure or character of the test substance. Therefore, in order to prove the result of the assay to be positive, it is necessary to confirm that the test substance is really an activator or an inhibitor to an enzyme by the other assay. However, the other assay with a complicated process may not be suitable for the first assay.
Recently, a high throughput screening (referred to as HTS) is very important on the early stage of the development of medicine. HTS enables several hundreds to ten thousands compounds to be assayed at the same time, but causes a problem of how to analyze an enormous number of data.
Journal of General Virology (2000), 81, 759–767 discloses that a pre-incubation of HCV RNA-dependent RNA polymerase in the presence of a template and a primer enhances an enzyme activity, without mentioning an assay of an activator or an inhibitor to an enzyme at all. The above document only discloses a character of the enzyme.
It is important to obtain useful information from data obtained by HTS without pseudo-positive data. A method for an assay of an activator or an inhibitor to an enzyme with no or little pseudo-positive data has been desired.